Method of producing 5-amino-4-imidaz-olecarboxamide ribofuranoside by fermentation



United States Patent 3,408,256 METHOD OF PRODUCING S-AMINO-4-IMEDAZ-OLECARBOXAMIDE RIBOFURANOSIDE BY FERlVIENTATION Hiroshi Sasaki and MikioAoki, Kanagawa-ken, Hircshi Okada and Kazumoto Kinoshita, Tokyo, andTeruo Shiro, Kanagawa-ken, Japan, assignors to Ajinomoto Co., Inc.,Tokyo, Japan No Drawing. Filed June 1, 1966, Ser. No. 554,305 Claimspriority, application Japan, June 8, 1965, 40/ 33,967 4 Claims. (Cl.195-28) The present invention relates to a method of producing5-amino-4-imidazolecarboxamide ribofuranoside (hereinafter referred toAICAR) by fermentation.

AICAR is being used as an intermediate for synthesizing components ofnucleic acid, which are valuable as seasonings, and is also a rawmaterial for synthesizing pharmaceuticals.

It is known that AICAR can be produced on an industrial scale byfermentative processes in which certain mutants of Bacillus subzilis orBacillus megutherium, are cultured on a nutrient medium containing acarbon source, a nitrogen source, and other nutrients, and AICAR isrecovered from the culture broth.

The known AICAR fermentation method is often interfered with byexcessive bacterial growth or metabolism and by spore formation.

We have found that AICAR fermentation can be improved by the presence ofsmall amounts of alkanoic acids in the culture medium, which controlspore formation and increase the yield of AICAR.

The alkanoic acids which can be used in the present invention have threeto nine carbon atoms in straight or branched chains and includepropionic acid, butyric acid,

caproic acid, and pelargonie acid. The minimum concentration of the acidwhich produces measurable beneficial eifects is determined by theprecision of the measuring method. The improvement is commensurate withthe amount of alkanoic acid at very low concentration, but reaches amaximum at a concentration of about 100 mg./dl. for butyric acid and atsimilar values for the other acids of the invention as far as they aresoluble. Some improvement is still observed at concentrations muchhigher than the optimum concentration.

Table 1 shows the effects of representative alkanoic acids on AICARfermentation. All tests were performed with a culture medium of thefollowing composition:

ml. batches of the medium were placed in 500 ml. shaking flasks andinoculated with Bacillus megatherizun MA-336 (ATCC No. 15117) andcultured at C. for 48 hours.

Bacterial growth was measured by the specific optical density of aspecimen diluted with water to 50 times its original volume to light ofwavelength 562 mu. Spore formation was evaluated by counting spores andcells in a microscopic field, and the percentage of spores wascalculated on the basis of the combined number of spores and cells. TheAICAR produced was determined in the culture broth by the method ofBratton-Marshal.

TABLE 1 Op. Spores, AICAR, Acid Mg/(ll. density Percent g./dl

Propionic 10 0. 76 20 0. S1 D0 30 O. 76 2 0. 93 50 0. 7S) 0 0. 93 10 0.80 50 0. 94. 30 0. 75 0 1. O1 50 0. 0 0. 9O 10 0. 84 0. 3 0. 95 20 0. 8G0 0. 15 0. 85 0 0. 85 5 0. 88 0 0. 90 1O 0. 87 0 O. 85 Pelargonie 2. 50. 87 0 0. Control 0 0. 80 17 0. 74

As is apparent from the table, butyric acid is superior to otheralkanoic acids in controlling spore formation and increasing the yieldof AICAR.

The soluble salts of alkanoic acids having 3 to 9 carbon atoms are aseffective as equivalent amounts of the corresponding free acids.

The composition of the culture medium is otherwise not critical and theusual carbon and nitrogen sources are employed.

The time after inoculation at which the alkanoic acid or its salt isadded to the culture medium has a distinct influence on the resultsobtained as is evident from the typical data on butyric acid (30mg./dl.) of Table 2. Specific optical density was determined (1) at thetime of butyric acid addition and (2) 48 hours after inoculation.Residual sugar was measured by the Lehmann-School method at the time ofbutyric acid addition only, and spores and AICAR were determined whenfermentation was terminated 48 hours after inoculation.

TABLE 2 Acid added Residual Optical density Spores, AICAR,

ter sugar, percent gJdl.

percent (1) As is shown in Table 2, the acid is preferably added to theculture medium not later than 20 hours after inoculation and while theresidual sugar present is at least 75% of the original value.

The bacteria whose production of AICAR benefit substantially from thepresent invention include Bacillus megatherium MA336 (ATCC No. 15117),Bacillus megatherium MA-'658 (ATCC No. 15118), Bacillus subtilis D422(ATCC No. 15115), and Bacillus subtilis D-25l1 (ATCC No. 15116). Allmembers of the family Bacillus which produce AICAR by fermentation werefound to give at least slightly better AICAR yields with the acids ofthis invention than without.

Mutants induced from the afore-menti-oned strains and lacking theability of spore formation produce AICAR in high yields in the presenceof the alkanoic acids and salts of this invention.

EXAMPLE 1 A seed culture was prepared on a culture medium containing 8g./dl. starch acid hydrolysate (glucose equivalent), 0.1 g./dl. KCl,0.04% MgSO .7H O, 2 ppm. each of Fe and Mn ion, 0.2 g./dl. caseinhydrolysate, 0.13 g./dl. yeast ribonucleic acid (containing 80% pureribonucleic acid), 1.5 g./ d1. NH Cl, and 5 g./dl. CaCO and sterilizedat C. for 10 minutes which was inoculated with a loopful of Bacillusmegatherium .MA-336 (ATCC No. 15117), and cultured at 30 C. for 20 hourswith shaking.

. .Fresh culture medium of the above composition was mixed with 30mg./dl. butyric acid, and 20 ml. batches of the mixed medium were placedin 500 m1. shaking flasks and sterilized at 115 C. for minutes. 0.1 ml.of the seed culture was added to each batch, and the culture wasincubated at 30 C. for 60 hours when the AICAR concentration in thebroth reached 1.2 g./dl.

A control batch without butyric acid produced 1.05 g./dl. underotherwise identical condition, and the fermentation was not completedafter 60 hours because of spore formation.

EXAMPLE 2 The procedure of Example 1 was repeated but starchhydrolysate, KCl and butyric acid were replaced by 8 g./dl. glucose,0.01 g./dl. KH PO and mg./dl. caproic acid, respectively. The culturedbroth contained 1.15 g./dl. AICAR after 60 hours.

40 to 45 percent spores formed in a control batch without caproic acid,the fermentation was not completed in 60 hours when the culture brothcontained only 0.95 g./dl. AICAR.

EXAMPLE 3 Bacillus subtilis D2511 (ATCC No. 15116) was inoculated on themedium of Example 1, cultured at C. for 60 hours, and AICAR was producedto a concentration of 0.6 g./dl.

A control batch without butyric acid contained 0.4 g./dl. AICAR, and thefermentation was not completed in 60 hours.

EXAMPLE 4 Seed cultures of Bacillus megatlzerium- MA-336 were preparedon eight 50 ml. batches of the medium of Example 1 in Sakaguchi flaskswhich were sterilized at 115 C. for 10 minutes, inoculated with aloopful of Bacillus megatherizun MA-336, and cultured until the specificoptical density reached 0.20.

Twenty liters of fermentation medium having the same composition as inExample 1 except for CaCO were poured into a liter fermentor, sterilizedat 110 C. for 10 minutes, and mixed with 6 g. of sterilized butyricacid. The medium was inoculated with the seed cultures and incubated at30 C. with stirring at 300 r.p.m. while aerating at A volume per minute.The pH of the medium was maintained within a range between 5.8 and 6.0by adding ammonia to the air. The fermentation was terminated whensamples showed no further increase of 7% residual sugar. A control testwithout butyric acid showed only 0.87 g./dl. AICAR and 18% residualsugar after hours. There were 50% spores in the control test, none inthe presence of butyric acid.

EXAMPLE 5 Culture media containing 8 g./dl. starch acid hydrolysate(glucose equivalent), 0.08 g./dl. KCl, 0.04 g./dl. MgSO .7H O, 2 p.p.m.of Fe and Mn ion, 0.09 g./dl. yeast ribonucleic acid, 1.7 g./dl. NH Cl,2.5 g./dl. CaCO and butyric acid as listed below were prepared, the pHof the media was adjusted to 7.2 with aqueous ammonia, and 20 ml.batches were placed in 500 ml. flasks.

After sterilizing at C. for 5 minutes, each medium was inoculated with aloopful of Bacillus megatherium 336-M-1 which had been induced fromBacillus megatherium MA336 (ATCC No. 15117) and had lost the ability ofspore formation, and cultured at 30 C. for 65 hours, whereupon theamount of AICAR produced was determined.

What we claim is:

1. In a process of producing 5-amino-4-imidazolecarboxamideribofuranoside by fermentation of an oqueous culture medium with abacterium of the genus Bacillus capable of producing S-amino 4imidazolecarboxa'mide ribofuranoside, the improvement which consists inadding to said culture medium an alkanoic acid having 3 to 9 carbonatoms or a salt of said acid which is soluble in said medium.

2. In a process as set forth in claim 1, said acid being butyric acid.

3. In a process as set forth in claim 2, the concentration of saidbutyricacid in said medium being'between l0 and 200 milligrams perdecileter.

4. In a process as set forth in claim 3, said bacterium being a memberof the species Bacillus megatherium and Bacillus subtilis.

References Cited UNITED STATES PATENTS 3,238,110 3/1966 Shiro et al. 28

ALVIN E. TANENHOL'IZ, Primary Examiner.

1. IN A PROCESS OF PRODUCING 5-AMINO-4-IMIDAZOLECARBOXAMIDERIBOFURANOSIDE BY FERMENTATION OF AN OQUEOUS CULTURE MEDIUM WITH ABACTERIUM OF THE GENUS BACILLUS CAPABLE OF PRODUCING5-AMINO-4-IMIDAZOLECARBOXAMIDE RIBOFURANOSIDE, THE IMPROVEMENT WHICHCONSISTS IN ADDING TO SAID CULTURE MEDIUM AN ALKANOIC ACID HAVING 3 TO 9CARBON ATOMS OR A SUIT OF SAID ACID WHICH IS SOLUBLE IN SAID MEDIUM.